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Background: Trypanosoma cruzi is subjected to hyperosmotic stress during its life cycle. Results: The recovery from hyperosmotic stress involves the function of an aquaporin, amino acid accumulation, polyphosphate synthesis, and global gene regulation. Conclusion:The response to hyperosmotic stress is different from that observed in mammalian cells or yeasts. Significance: Learning the mechanism of osmoregulation is important for finding new drug targets.

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that Ͼ90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.

Epimastigotes of Trypanosoma cruzi (Tulahuen strain Tul 0 stock) biotransform benznidazole (N-benzyl-2-nitro-1-imidazole acetamide) to reactive metabolites that bind covalently to DNA, proteins and lipids of the parasite. These effects might be related to the trypanocidal action of benznidazole, a chemotherapeutic agent against Chagas' disease.

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).

Cells of the insect parasite Crithidia fasciculata incubated with ['4C]glucose were found to possess only one lipidbound oligosaccharide with solubility in chloroform/methanol/ water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Mana-+Mana---Mana--[Mana-+ Mana-+Man(al--6)]Man--GlcNAc(l1--4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-13-N-acetylglucosaminidase H. One ofthe protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation ofproteins in the protozoan Crithidiafasciculata does not involve glucosylated lipid-bound oligosaccharides as intermediates. It has become evident in recent years that glycosylation of asparagine residues in eukaryotic cell proteins involves dolichol pyrophosphate (DolPP)-bound oligosaccharides as intermediates (1). It has been reported that in animal tissues (2, 3), yeasts (4-6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9). However, no evidence was presented indicating that the oligosaccharides used in the assay had the same specific activity.The protein-bound oligosaccharides are then processed by loss of some of their monosaccharide constituents and addition ofother residues directly from the respective sugar nucleotides. We here report evidence suggesting that in the protozoan Crithidta fasciculata, an insect obligate parasite, the oligosaccharide transferred to protein contains two N-acetylglucosamine and seven mannose residues and that the oligosaccharide may be processed once bound to protein.MATERIALS AND METHODS Materials.[14C]Glucose (284 Ci/mol; 1 Ci = 3.7 X 1010 becquerels) was from New England Nuclear. Jack bean a-mannosidase type III and Streptomyces griseus protease type VI were purchased from Sigma and endo-,B3N-acetylglucosaminidase H (endo H), from Miles. Isolation of Lipid-Bound Oligosaccharides. C. fasciculata cells (Anopheles isolate ATCC 11745) were grown in the medium described by Bacchi et aL (10) without agar at 28°C. Six hundred milliliters of culture containing about 2 g of cells (logarithmic phase) were cooled on ice and centrifuged at 2500 X g for 10 min at 4°C. The cell pellet was resuspended in 30 ml of ice-cold minimal Eagle's medium without glucose but containing 5...

Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH2-OCO-(2,4,6-Me3)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi. The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 microM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi, and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.

N-linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc1Man5-9GlcNAc2 and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man9GlcNAc2 in protein N-glycosylation) were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. About 52-53% total N-linked oligosaccharides appeared to have glucose residues. The compounds were identified as Glc1Man7-9GlcNAc2. The same percentage was obtained when cells were pulsed-chased with [14C]glucose in the presence of deoxynojirimycin for 60 min. No evidence for the presence of an endomannosidase yielding GlcMan from the glycosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-53% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.